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| United States Patent |
5,759,571
|
|
Hersch
, et al.
|
June 2, 1998
|
Antibiotic formulation and use for drug resistant infections
Abstract
A liposomal aminoglycoside formulation comprising a neutral lipid, a
negatively charged lipids and a sterol. The formulation contains
unilamellar vesicles having an average size below 100 nm. A process of
making liposomes containing an aminoglycoside is provided where the
hydration temperature is significantly below the transition temperature of
the formulation. A method for the treatment of drug susceptible and drug
resistant bacteria.
| Inventors:
|
Hersch; Evan M. (Tucson, AZ);
Petersen; Eskild A. (Tucson, AZ);
Proffitt; Richard T. (Arcadia, CA);
Bracken; Kevin R. (Sunland, CA);
Chiang; Su-Ming (Canoga park, CA)
|
| Assignee:
|
NeXstar Pharmaceuticals, Inc. (Boulder, CO)
|
| Appl. No.:
|
084218 |
| Filed:
|
June 23, 1993 |
| PCT Filed:
|
May 11, 1993
|
| PCT NO:
|
PCT/US93/04501
|
| 371 Date:
|
June 23, 1993
|
| 102(e) Date:
|
June 23, 1993
|
| Current U.S. Class: |
424/450 |
| Intern'l Class: |
A61K 009/127; A61K 009/133 |
| Field of Search: |
424/450
264/4.1,4.3,4.6
|
References Cited
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|
| 4522803 | Jun., 1985 | Lenk et al. | 424/1.
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| 4621023 | Nov., 1986 | Redziniak et al. | 428/402.
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| 4753788 | Jun., 1988 | Gamble | 424/1.
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| 4897384 | Jan., 1990 | Janoff et al. | 514/34.
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| 4933121 | Jun., 1990 | Law et al. | 264/43.
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| 4946683 | Aug., 1990 | Forssen | 424/422.
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| 4981692 | Jan., 1991 | Popescu et al. | 424/422.
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| Foreign Patent Documents |
| 0087993 | Jan., 1990 | EP.
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| 0500143 | Aug., 1992 | EP.
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| 0565361 | Jul., 1996 | EP.
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| 7082311 | May., 1982 | JP.
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| WO 85/00751 | Feb., 1985 | WO.
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| WO 88/04573 | Jun., 1988 | WO.
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| WO 85/00515 | Feb., 1989 | WO.
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| 9323015 | Nov., 1993 | WO.
| |
Other References
Bakker-Woudenberg et al., Efficacy of gentamicin of ceftazidime entrapped
in liposomes with prolonged blood circulation and enhanced localization in
Klebsiella pneumoniae-Infected lung tissue, J. Infect. Dis. 171:938-947,
1995.
Bermudez et al., "Treatment of disseminated Mycobacterium avium complex
infection of beige mice with liposome-encapsulated aminoglycosides," J.
Infect. Dis. 161:1262-1268, 1990.
Bermudez et al., "Intracellular killing of Mycobacterium avium complex by
rifapentine and liposome-encapsulated amikacin," J. Infect. Dis.
156(3):510-513, 1987.
Bonventre & Gregoriadis, "Killing of intraphagocytic staphylococcus aureus
by dihydrostreptomycin entrapped within liposomes," Antimicrobial Agents
and Chemotherapy 13(6):1049-1051, 1978.
Gangadharam et al., "Comparative activities of free and liposome
encapsulated amikacin against mycobacterium avium complex (MAC)," Lipsomes
in the Therapy of Infectious Diseases and Cancer, pp. 177-190, Alan R.
Liss, Inc., 1989.
Guzgunes et al., "Treatment of mycobacterium avium-intracellulare complex
infection in beige mice with free and liposome-encapsulated streptomycin:
Role of liposome type and duration of treatment," J. Infect. Dis.
164:143-151, 1991.
Karlowsky and Zhanel, "Concepts on the use of liposomal antimicrobial
agents: Applications for aminoglycosides," Clinical Infect. Dis.
15:654-667, 1992.
Majumdar et al., "Efficacies of liposome-encapsulated streptomycin and
ciprofloxacin against mycobacterium avium-M, intracellulare complex
infections in human peripheral blood monocyte/macrophages," Antimicrobial
Agents & Chemotherapy 36(12):2808-2815, 1992.
Saito and Tomioka, "Therapeutic efficacy of liposome-entrapped rifampin
against mycobacterium avium complex infection induced in mice,"
Antimicrobial Agents & Chemotherapy 33(4):429-433, 1989.
Schreier et al., "Sustained release of liposome-encapsulated gentamicin and
fate of phospholipid following intramuscular injection in mice," J.
Controlled Release 5:187-192, 1987.
Stevenson et al., "Enhanced activity of streptomycin and chloramphenicol
against intracellular escherichia coli in the J774 macrophage cell line
mediated by liposome delivery," Antimicrobial Agents & Chemotherapy
24(5):742-749, 1983.
Tadakuma et al., "Treatment of experimental salmonellosis in mice with
streptomycin entrapped in liposomes," Antimicrobial Agents & Chemotherapy
28(1):28-32, 1985.
Abstract: Wichert, el al., "Characterization, aerosolization, and in vitro
activity against Mycobacterium avium-intracellulare in alveolar
macrophages," Int. J. Pharm., 78/2-3:227-235 (1992).
Fountain, Michael et al., Liposome-Cell Interactions a Rapid Assay For
Cells in Suspension Cultu Biochimica et Biophysica Acta, 596:420-425
(1980).
Dees, C. et al., "Enhanced Intraphagocytic Killing of Brucella Abortus in
Bovine Monouclear Cells Liposomes-Containing Gentamicin." Veterinary
Immunology and Immunopathology, vol. 8:171-18 (1985).
Barza, Michael et al., "Pharmacokinetics of Subconjunctival
Liposome-Encapsulated Gentamicin i Normal Rabbit Eyes." Investigative
Ophthalmology and Visual Science, vol. 25:486-490, (Apr. 19.
Fishman, Peggy, et al., "Intravitreal Liposomes-Encapsulated Gentamicin in
a Rabbit Model." Investigative Ophthalmology and Visual Science, vol.
27:1103-1106 (Jul. 1986).
Kubo, Masaharu, et al., "Binding of Propranolo and Gentamicin to Small
Unilamellar Phospholipid Vesicles." Biochemical Pharmacology, vol. 35, No.
21:3761-3765 (1986).
Gabizon, A., "Liposome as in Vivo carriers of Adrizmycin: Reduced cardiac
uptake and preserved antitumor activity in mice", Cancer Research 42, Nov.
1982, pp. 4734-4739.
Crommelin, D.J.A., "Preparation and characterization of
doxorubicin-containing liposomes: I. Influence of liposome charge and pH
of hydration medium on loading capacity and particle size", International
Journal of Pharmaceutics, 16, (1983), pp. 79-92.
Rosa, P, "Liposomes Containing Doxorubicin: An example of drug targeting",
Transport in Biomembranes, 1982, p. 243.
International Search Report dated 22 Sep. 1993.
Dialog Information Services, File 73 Embase, Dialog accession No. 8333576,
Embase accession No. 92010722 Wichert B.V. et al.
Fountain, Michael W., et al., "Enhanced Intracellular Killing of
Staphylococcus Aureus by Canine Monocytes Treated with Liposomes
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Microbiology, vol. 6:373-376 (1981).
|
Primary Examiner: Kishore; Gollamudi S.
Attorney, Agent or Firm: NeXstar Pharmaceuticals, Inc.
Claims
What is claimed:
1. A composition for treating a bacterial infection in a patient consisting
essentially of an aminoglycoside, encapsulated in liposomes, wherein the
liposomes are comprised of cholesterol, a neutral amphiphilic lipid and a
negatively charged amphiphilic lipid.
2. The composition of claim 1 wherein the neutral and negatively charged
amphiphilic lipids are saturated.
3. The composition of claim 2 wherein the saturated neutral and negatively
charged amphiphilic lipids are phospholipids.
4. The composition of claim 3 wherein the saturated neutral phospholipid is
selected from the group consisting of hydrogenated egg phosphatidylcholine
(HEPC), dimyristoylphosphatidylcholine (DMPC), hydrogenated soy
phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC), and
dipalmitoyl phosphatidylcholine (DPPC).
5. The composition of claim 3 wherein the saturated neutral phospholipid is
hydrogenated soy phosphatidylcholine (HSPC).
6. The composition of claim 3 wherein the saturated negatively charged
phospholipid is selected from the group consisting of hydrogenated soy
phosphatidylglycerol (HSPG), hydrogenated egg phosphatidylglycerol (HEPG),
distearyolphosphatidylglycerol (DSPG), dimyristoyl phosphatidylglycerol
(DMPG), and dilaurylphosphatidylglycerol (DLPG).
7. The composition of claim 3 wherein the saturated negatively charged
lipid is distearoylphosphatidylglycerol (DSPG).
8. The composition of claim 1 wherein said aminoglycoside is selected from
the group consisting of streptomycin, neomycin, kanamycin, gentamicin,
tobramycin, sisomicin, amikacin, and netilmicin.
9. The composition of claim 1 wherein said aminoglycoside is amikacin.
10. A composition for inhibiting bacterial growth in a patient consisting
essentially of an aminoglycoside encapsulated in liposomes, wherein the
liposomes are comprised of cholesterol, a neutral amphiphilic lipid and a
negatively charged amphiphilic lipid, wherein the neutral amphiphilic
lipid, cholesterol and negatively charged amphiphilic lipid are in a molar
ratio of about 2:1:0.1, wherein the aminoglycoside to total lipid molar
ratio is from 1:9 to 1:3 and wherein said liposomes consist of unilamellar
vesicles having an average size of less than 100 nm.
11. A composition for treating a bacterial infection in a patient
consisting essentially of amikacin encapsulated in liposomes, wherein the
liposomes are comprised of cholesterol, HSPC, and DSPG wherein
HSPC:cholesterol:DSPG are in a molar ratio of about 2:1:0.1 wherein the
amikacin to total lipid molar ratio is from 1:9 to 1:3 and wherein said
liposomes consist of unilamellar vesicles having an average size of less
than 100 nm.
Description
This is a 371 of PCT/US93/04501, filed May 11, 1993, which is a
Continuation-in-Part application of PCT/US92/10591, filed Dec. 2, 1992.
FIELD OF THE INVENTION
This invention relates to the fields of biochemistry and medicine, and
particularly to a liposome formulation. More specifically, it relates to a
liposomal formulation containing an aminoglycoside, its process of
manufacture and its use. This invention also relates to formulations
having reduced toxicity, longer stability, and superior efficacy. This
invention further relates to liposomal formulations containing amikacin
and its use in treating drug susceptible and drug resistant strains of
bacterial infections.
BACKGROUND OF THE INVENTION
The discovery of aminoglycosides began in the 1940s with the isolation of
streptomycin from Streptomyces griseus. Since the 1940s, other
aminoglycosides have been discovered and synthesized. These include
neomycin which is obtained or isolated from Streptomyces fradiae;
kanamycin which is isolated from Streptomyces kanamyceticus; gentamicin
which is isolated from Micromonospora purpurea; tobramycin which is
isolated from Streptomyces tenedrarius; sisomicin isolated from
micromonospora inyoesis; amikacin which is a semisynthetic derivative of
Kanamycin A; and netilmicin which is a semisynthetic derivative of
sisomicin. Amikacin has the broadest spectrum of antimicrobial activity of
all the aminoglycosides. It also has a unique resistance to the
immunoglycoside-inactivating enzymes.
The aminoglycosides are polar-cations which consist of two or more amino
sugars joined in a glycosidic linkage to a hexose nucleus, which is
usually in a central position. The aminoglycosides are used primarily to
treat infections caused by gram-negative bacteria. However,
aminoglycosides have been used in recent years to treat bacteria from the
genera Mycobacteria. For example, amikacin has shown to be effective
against Mycobacterium tuberculosis. Aminoglycosides have also been tested
against M.avium infections including M. avium-intracellulare complex (MAC)
which is a group of related acid-fast organisms that grow only slightly
faster than M. tuberculosis and can be divided into a number of serotypes.
At the beginning of the twentieth century, tuberculosis was the most
prevalent cause of death in the United States. By the late 1940s, with the
advent of streptomycin, tuberculosis infection had decreased
substantially. Since the mid-1980s with the appearance of the acquired
immune deficiency syndrome, tuberculosis again began to emerge as a major
health problem. Further, the new cases of tuberculosis showed resistance
to many of the available antibiotic therapies. Similarly MAC, once
considered rare, is now the most common systemic bacterial type infections
in patients suffering from acquired immune deficiency syndrome. Hence, the
search for an effective antibiotic has intensified.
Although the aminoglycosides have been useful in treating infections, the
use of these antibiotics is not free from toxicity and side effects.
Aminoglycosides may produce irreversible vestibular, cochlear, and renal
toxicity. The two main toxic effects of aminoglycosides are ototoxicity
and nephrotoxicity. Studies have found that the aminoglycosides
antibiotics may cause polyuria, decreased urinary osmolality, proteinuria,
enzymuria, glycosuria, and a decrease in the rate of glomerular
filtration. Some investigators believe that nephrotoxicity results from
the accumulation of the aminoglycosides in the renal cortex because of the
long half-life of the agents in that tissue.
Liposomes are microscopic vesicles made, in part, from phospholipids which
form closed, fluid filled spheres when dispersed with water. Phospholipid
molecules are polar, having a hydrophilic ionizable head and two
hydrophobic tails consisting of long fatty acid chains. Thus, when
sufficient phospholipid molecules are present with water, the tails
spontaneously associate to exclude water while the hydrophilic phosphate
heads interact with water. The result is a bilayer membrane in which the
fatty acid tails converge in the newly formed membrane's interior and the
polar heads point in opposite directions toward an aqueous medium. These
bilayer membranes can be caused to form closed spheres known as liposomes.
The polar heads at the inner surface of the membrane point toward the
aqueous interior of the liposome. At the opposite surface of the spherical
membrane, the polar heads interact with the surrounding aqueous medium. As
the liposomes are formed, water soluble molecules can be incorporated into
the aqueous interior, and lipophilic molecules will tend to be
incorporated into the lipid bilayer. Liposomes may be either
multilamellar, like an onion with liquid separating many lipid bilayers,
or unilamellar, with a single bilayer surrounding an entirely liquid
center.
There are many types of liposome preparation techniques which may be
employed and which produce various types of liposomes. These can be
selected depending on the use, the chemical intended to be entrapped, and
the type of lipids used to form the bilayer membrane. The requirements
which must be considered in producing a liposome preparation are similar
to those of other controlled release mechanisms. They are as follows: (1)
high percent of chemical entrapment; (2) increased chemical stability; (3)
low chemical toxicity; (4) rapid method of production; and (5)
reproducible size distribution.
The first method described to encapsulate chemicals in liposomes involved
production of multilamellar vesicles (MLVs). Methods for encapsulating
chemicals in MLVs are known in the art.
Liposomes can also be formed as unilamellar vesicles (UVs), which generally
have a size less than 1 .mu.m. There are several techniques known in the
art which are used to produce unilamellar liposomes.
Smaller unilamellar vesicles can be formed using a variety of techniques.
By dissolving phospholipids in ethanol and injecting them into a buffer,
the lipids will spontaneously rearrange into unilamellar vesicles. This
provides a simple method to produce UVs which have internal volumes
similar to that of those produced by sonication (0.2-0.5 L/mol/lipid).
Sonication or extrusion (through filters) of MLVs also results in
dispersions of UVs having diameters of up to 0.2 .mu.m, which appear as
clear or translucent suspensions.
Another common method for producing small UVs is the detergent removal
technique. Phospholipids are solubilized in either ionic or non-ionic
detergents such as cholates, Triton X, or n-alkylglucosides. The drug is
then mixed with the solubilized lipid-detergent micelles. Detergent is
then removed by one of several techniques: dialysis, gel filtration,
affinity chromatography, centrifugation, ultrafiltration. The size
distribution and entrapment efficiencies of the UVs produced this way will
vary depending on the details of the technique used.
The therapeutic use of liposomes includes the delivery of drugs which are
normally toxic in the free form. In the liposomal form the toxic drug may
be directed away from the sensitive tissue and targeted to selected areas.
Liposomes can also be used therapeutically to release drugs, over a
prolonged period of time, reducing the frequency of administration. In
addition, liposomes can provide a method for forming an aqueous dispersion
of hydrophobic drugs for intravenous delivery.
When liposomes are used to target encapsulated drugs to selected host
tissues, and away from sensitive tissues, several techniques can be
employed. These procedures involve manipulating the size of the liposomes,
their net surface charge as well as the route of administration. More
specific manipulations have included labeling the liposomes with receptors
or antibodies for particular sites in the body.
The route of delivery of liposomes can also affect their distribution in
the body. Passive delivery of liposomes involves the use of various routes
of administration, e.g., intravenous, subcutaneous and topical. Each route
produces differences in localization of the liposomes. Two common methods
used to actively direct the liposomes to selected target areas are binding
either antibodies or specific receptor ligands to the surface of the
liposomes. Antibodies are known to have a high specificity for their
corresponding antigen and have been shown to be capable of being bound to
the surface of liposomes, thus increasing the target specificity of the
liposome encapsulated drug.
Since the chemical composition of many drugs precludes their intravenous
administration, liposomes can be very useful in adapting these drugs for
intravenous delivery. Many hydrophobic drugs, including cyclosporine, fall
into this category because they cannot be easily dissolved in a
water-based medium and must be dissolved in alcohols or surfactants which
have been shown to cause toxic reactions in vivo. Liposomes, composed of
lipids, with or without cholesterol, are nontoxic. Furthermore, since
liposomes are made up of amphipathic molecules, they can entrap
hydrophilic drugs in their interior space and hydrophobic molecules in
their lipid bilayer. Although methods for making liposomes are well known
in the art, it is not always possible to determine a working formulation
without undue experimentation.
Liposomal formulations containing aminoglycosides have been prepared. Many
of the preparations include aerosol formulations using MLVs. Other
formulations contain a large amount of negatively charged lipids,
generally is greater than 20%, to increase retention time or circulation
half-life. Problems associated with aminoglycosides liposomal formulations
include short retention time in the system because of RES uptake.
Attempted formulations in the art have also resulted in liposomal
aminoglycosides that are unstable--both on the shelf and in serum.
Thus, it is a desideratum to provide for a novel formulation which would
increase the retention time of an aminoglycoside in a mammal's system and
which can deliver more drug with superior efficacy and lower toxicity then
free drug. It is also desirable to provide a process which allows the
manufacture of a clear, stable and efficacious aminoglycoside liposome
suspension because of the inability of those of ordinary skill to produce
a therapeutically effective aminoglycoside unilamellar liposomal
formulation having an average particle size of less than 100 nm,
preferably with a high drug to total lipid ratio.
SUMMARY OF THE INVENTION
Liposomes are provided in the present invention which comprise an
aminoglycoside wherein the liposomes are unilamellar having an average
size of 100 nm or less. A liposomal formulation is provided which
comprises an aminoglycoside wherein the liposomes are comprised of a
neutral lipid, a sterol and a negatively charged lipid. Small unilamellar
vesicles are also provided wherein the molar amount of negatively charged
lipid is less than 20% of total lipid. A preferred formulation is
liposomes having an aminoglycoside wherein the liposomes are unilamellar
vesicles having an average size of 30 nm to 100 nm and further comprised
of a phosphatidylcholine, cholesterol, and a phosphatidylglycerol wherein
the molar amount of phosphatidylglycerol is less than 5% and preferably
about 3%. The drug to total lipid ratio is between 1:9 and 1:3 with the
preferred ratio at about 1:4. A preferred formulation is also provided
comprising liposomes including an aminoglycoside wherein the liposomes are
unilamellar vesicles having an average size less than 100 nm wherein the
lipids comprise hydrogenated soy phosphatidylcholine, cholesterol, and
distearoylphosphatidylglycerol in a molar ratio of about 2:1:0.1.
The present invention also includes a method for making aminoglycoside
liposomes. The process comprises forming a lipid powder comprised of a
neutral phospholipid, a sterol, and a negatively charged lipid; mixing the
powder with an aminoglycoside in an aqueous buffer and hydrating the
mixture at a temperature significantly below the transition temperature of
the lipid mixture; and reducing the size of the liposome to an average
particle size of less than 100 nm.
The present invention also provides for the treatment of bacterial
infections in mammals comprising preparing a liposomal formulation having
an aminoglycoside wherein the liposomes are those described above and
which are used to treat infections by introducing a therapeutic effective
amount of the liposomes into a mammal. Thus, the present invention
provides the use of liposomal aminoglycoside formulations to treat
bacterial infections. The bacterial infections treated include
opportunistic aerobic gram-negative bacilli such as the genera
Pseudomonas. Another aspect of the invention includes the use of the
liposomal formulations in the treatment of a bacterial infection caused by
P. aeruginosa. The method of treating bacterial infections is not limited
to gram-negative infections. The liposomal formulations can be used to
treat bacterial infections comprising gram-positive bacilli such as the
genera Mycobacterium. The invention is particularly useful for treating
mycobacterium which causes tuberculosis-like diseases. Numerous bacterium
may be treated using the liposomes described above. The bacteria would
include: M. tuberculosis, M. leprae, M. Intracellulare, M. smegmatis, M.
bovis, M. kansasii, M. avium, M. scrofulcium, or M. africanum. Liposomal
formulations of aminoglycoside are also particularly useful in treating
MAC.
A particularly useful aspect of the invention is a method of treating a
drug resistant bacterial infection in a patient, comprising the delivery
to the patient an effective amount of liposomes comprising an encapsulated
aminoglycoside wherein the liposomes are comprised of unilamellar vesicles
comprised of a neutral lipid, cholesterol and a negatively charged lipid,
and having an average size of less than 100 nm. Experiments performed in
vitro establish that liposomal amikacin inhibits and kills drug resistant
M. tuberculosis. The experiments performed further establish that
liposomal amikacin kills M. tuberculosis whereas the free drug, at
equivalent dosage concentration, only inhibits the growth of the bacteria.
Killing is defined as a reduction in the number of colony forming units of
bacteria from a previous time point. Inhibition is defined as an increase
in, or the same number of, colony forming units of bacteria from a
previous time point but less than the number of colony forming units shown
for untreated cultures at the same time points. Thus, the present
invention provides for the killing of the bacteria at tolerable non-toxic
levels in cases where the bacteria is resistant to aminoglycosides and
other antibiotics or where the free drug has at the most an inhibitory
effect.
The present invention also shows that liposomal amikacin is retained in
blood plasma significantly longer than free amikacin. Intermittent
treatment of non-compliant patients is obtained in the present invention
as the present invention provides higher peak serum levels, prolonged
serum half life and increased uptake and retention by macrophages.
The present invention also provides a method for the treatment of drug
susceptible M. tuberculosis by delivering an effective amount of liposomal
amikacin to a patient wherein the dosage levels provide inhibition or
killing at levels equivalent to or greater than free amikacin. Thus
advantages provided by the decreased toxicity and increased serum levels
of amikacin delivered by liposomal amikacin provide a preferable and
useful alternative to treatment provided by the free (unencapsulated)
amikacin.
Many, if not all, mycobacterium infections discussed above are difficult to
treat because the bacteria invade phagocytic cells such as macrophages.
Application of liposomal formulations containing an aminoglycoside result
in the intracellular delivery of the drug which would not normally occur
with the delivery of the free drug. Thus, the present invention provides a
treatment of infected phagocytic cells in mammals by delivering a
therapeutic or effective amount of an aminoglycoside into a macrophage
using a unilamellar liposome having an average size of 100 nm or less.
Provided herein is a liposomal amikacin formulation which can be delivered
to a mammal and which provides the following benefits over free amikacin:
1) significantly higher doses of amikacin delivered to sites of infection;
2) substantially lower toxicity; and 3) retention in blood plasma for a
significantly longer period of time.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term liposome refers to unilamellar vesicles or
multilamellar vesicles such as described in U.S. Pat. Nos. 4,753,788 and
4,935,171, the contents of which are incorporated herein by reference.
The present invention provides liposomal aminoglycoside formulation
preferably containing a neutral lipid such as a phosphatidylcholine, a
phosphatidylglycerol, cholesterol (CHOL) and amikacin. Preferred lipid
include lipids which are chemically pure and/or are fully saturated. The
preferred neutral lipids are saturated lipids such as hydrogenated egg
phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC),
distearoyl phosphatidylcholine (DSPC), and dipalmitoyl phosphatidylcholine
(DPPC). The preferred carbon chain lengths of the neutral lipids are from
C.sub.16 -C.sub.18. The preferred negatively charged lipids are saturated
lipids such as hydrogenated soy phosphatdylglycerol (HSPG), hydrogenated
egg phosphatidylglycerol (HEPG), distearyolphosphatidylglycerol (DSPG),
dimyristoylphosphatidylcholine. Hydrogenated soy phosphatidylcholine
(HSPC), distearoylphosphatidylglycerol (DSPG) are the preferred lipids for
use in the invention. Other suitable phosphatidylcholines include those
obtained from egg or plant sources, or those that are partially or wholly
synthetic. Other phosphatidylglycerols that may be used are saturated
semisynthic lipids having carbon chain lengths from C.sub.12 -C.sub.18 and
include dimyristoyl phosphatidylglycerol (DMPG) and
dilaurylphosphatidylglycerol (DLPG).
The preferred formulation includes liposomes comprising an encapsulated
aminoglycoside wherein the liposomes are unilamellar vesicles having an
average size of less than 100 nm and which are stable for at least two
weeks at 22.degree. C. without a significant change in size or without
loss of more than 10% of encapsulated aminoglycosides. Generally the size
of the liposomes would not vary by more than 30% and most preferably would
not vary by more than 20%. The preferred ratio of HSPC:CHOL:DSPG is about
2:1:0.1 and the drug to total lipid ratio is about 1:4. Other preferred
formulations include DSPG in a molar amount of 0 to 20% and most
preferably in a molar amount of less than 5%. Other preferred formulations
include formulations where the drug to total lipid ratio is from 1:9 to
1:3.
The process of the present invention is initiated with the preparation of a
solution from which the liposomes are formed. A quantity of a
phosphatidylcholine, a phosphatidylglycerol and cholesterol is dissolved
in an organic solvent, preferably a mixture a 1:1 (by volume) mixture of
chloroform and methanol, to form a clear solution. Other solvents (and
mixture thereof), such as ether, ethanol and other alcohols can be used.
The preferred temperature to dissolve the lipids is between room
temperature and 60.degree. C., preferably at room temperature. The
solution is evaporated to form a lipid film or a lipid powder. To form a
lipid film, the solvents are evaporated under nitrogen between room
temperature and 60.degree. C., preferably at room temperature. To form a
lipid powder, the mixture of lipids in solution as described above is
sprayed in a spray drier. Preferably, the spraying takes place under
nitrogen.
An aminoglycoside, for example, amikacin free base, is dissolved in an
aqueous phosphate buffer with 9% sucrose and the pH is adjusted between 6
and to 8, preferably between 6 and 7.5 and most preferably between 6.2 and
6.6. The preferred pH is about 6.4. The buffer may also be adjusted to a
pH of about 7.4. The pH of the buffer is adjusted with diluted acids and
bases preferably with 6N HCI and 2.5N NaOH. The preferred buffer is 10 mM
phosphate buffer. However, other aqueous buffers can be used such as
succinate buffer (Disodium succinate hexahydrate). The aminoglycoside
solution is mixed with either the lipid film or the lipid powder and
hydrated, preferably between 40.degree. C. and 65.degree. C. most
preferably between 45.degree. C. and 65.degree. C. The solution should be
hydrated for at least ten minutes.
Unilamellar vesicles are formed by the application of a shearing force to
the hydrated solution, e.g., by extrusion, sonication, or the use of a
homogenizing apparatus such as a Gaulin homogenizer or a French press.
Shearing force can also be applied using injection, freezing and thawing,
dialyzing away a detergent solution from lipids, or other known methods
used to prepare liposomes. The size of the liposomes can be controlled
using a variety of known techniques including the duration of shearing
force. Preferably, the modified Gaulin homogenizing apparatus described in
U.S. Pat. No. 4,753,788 is employed to form unilamellar vesicles having
diameters of less than 100 nm at a pressure of 7,000 to 13,000 psi and a
temperature significantly below the transition temperature of the lipids.
The above described formulations are particularly useful for the treatment
of MAC and Pseudomonas aeruginosa infections. The use of the above
formulations indicate that MAC may be treated with significantly more
amikacin delivered in a liposomal formulation than with free drug. For
example it has been shown that up to 320 mg of amikacin per kilogram of
mice body-weight which is more than 50% more than is tolerated with free
drug. The use of the above formulations also indicates that a liposomal
formulation is able to deliver significantly more amikacin to a mammal
than free drug in the treatment of P. aeruginosa.
Amikacin delivered through a liposomal formulation is also retained in the
plasma longer than free amikacin.
The above described formulations are also efficacious in inhibiting and
killing both drug resistant and drug susceptible M. tuberculosis as
established by in vitro testing. In one experiment the drug resistant
strain Vertulla of M. tuberculosis was tested. In another experiment the
drug susceptible strain H37RV was tested. The experiments were carried out
as described in Example 6. Briefly, human monocytes derived macrophage
cultures were developed and infected with either the drug resistant strain
or the drug susceptible strain. Liposomal amikacin was prepared as
described in Example 1 below. The liposomes comprised HSPC, cholesterol
and DMPG in a molar ration of about 2:1:0.1. The drug to total lipid ratio
was 1:4 (about 25%). The average size of the liposomes was under 100 nm.
In the experiment performed on the susceptible strain, liposomal and free
amikacin were added to the cultures at the following concentrations
(.mu.g/ml): 1, 2, and 4. Untreated cultures and cultures treated with
liposomes-only were used as controls. The cultures were assayed at 0, 4,
and 7 days after infection. The 1 .mu.g/ml concentrations of both the free
amikacin and the liposomal amikacin inhibited the growth of the bacteria
with the liposomal amikacin showing greater inhibition and also killing at
day 4. The 2 .mu.g/ml concentrations of both the free amikacin and the
liposomal amikacin were also effective in treating the infection wherein
the liposomal amikacin obtained killing at day 4 and the free drug
obtained inhibition. The 4 .mu.g/ml concentrations of both the liposomal
and the free amikacin obtained killing at day 4 with the liposomal
amikacin showing greater killing. Thus, liposomal amikacin provides a
preferred treatment of M. tuberculosis because the present invention
provides, at the least, equivalent inhibition and killing at similar
concentrations than free amikacin with lower toxicity and longer plasma
retention time.
In the experiment performed on the resistant strain, liposomal and free
amikacin were added to the cultures at the following concentrations
(.mu.g/ml): 4, 8, and 16. Untreated cultures and cultures treated with
liposomes-only were used as controls. The cultures were assayed at 0, 4,
and 7 days after infection. At day 4 the liposomal amikacin achieved a
bacterial growth of a least twenty-one-fold, sixty-four-fold and several
hundred-fold (e.g. 563.times.) less than the untreated control for the 4
.mu.g/ml, 8 .mu.g/ml and 16 .mu.g/ml concentration, respectively. This
activity was 8.7, 12.9 and 130 times more effective than for the
respective free amikacin concentrations.
At day 7 the liposomal amikacin achieved bacterial growth of at least
one-hundred-fold, (e.g. 133.times.), one-thousand-fold (e.g. 1110.times.)
and several thousand-fold (e.g. 5880.times.) less than the untreated
control. This activity was 41, 11.9 and 46 times more effective than for
the respective amikacin concentrations.
The results show that free amikacin would only inhibit the growth of the
infection without killing at the concentrations tested. The liposomal
amikacin provided killing of the drug resistant bacteria at all
concentrations at day 4. Thus, liposomal amikacin provided high degree of
killing where only an inhibitory effect was expected against the drug
resistant strain.
Since dosage regimens for aminoglycosides are well known to medical
practitioners, the amount of the liposomal aminoglycoside formulation
which is effective for the treatment of infections in mammals and
particularly humans will be apparent to those skilled in the art.
This invention will be more fully understood by reference to the following
examples, which are intended to be illustrative of the invention, but not
limiting thereof. Examples 1-6 detail the formation, and both chemical and
biological testing of the liposomal amikacin formulations of the
invention.
EXAMPLE 1
A lipid mixture of hydrogenated soy PC, cholesterol, and distearoyl
phosphatidylglycerol was provided in a molar ratio of 2:1:0.1
respectively. The lipids were dissolved in chloroform and methanol (1:1 by
volume). The resulting solution was stirred until the lipids dissolved and
a clear solution was formed. The mixing is best carried out at room
temperature. A lipid film was obtained by evaporating the organic solvents
under Nitrogen at room temperature.
Amikacin free base was dissolved in 10 mM phosphate buffer with 9% sucrose
and the pH adjusted to 7.4 with 6.0M HCl. The lipid film and the amikacin
solution were mixed so that the concentration of the drug was about 250
mg/ml and the concentration of the lipid was about 100 mg/ml. The
resulting solution was stirred and hydrated for 10-15 minutes at
65.degree. C. The solution was sonicated for 20 minutes using a probe
sonicator (Model 500 Sonic & Material). The solution was held at
65.degree. C. for ten minutes. The solution was cooled to room temperature
and centrifuged at 3600 rpm for 10 minutes. The supernatant was collected
and poured through a Sephadex G-50 column to separate the liposomal
formulation from the free drug. The concentration of amikacin and lipid
components were determined by HPLC assay. The size was determined by
optical particle sizing. The size of the liposomes varied from experiment
to experiment from about 40-100 nm. For example, in one experiment a mean
diameter of 62.1 nm was observed. In another, a mean diameter of 47.4 nm
was observed. A preferred formulation contains a drug to lipid ratio of
1:4.
EXAMPLE 2
A lipid solution was prepared in methanol and chloroform as described in
Example 1. A lipid powder was obtained in a spray drier (Yamato Pulvis
Basic Unit, Model GB-21). The following settings were used: 1) pump dial
at 3-4.5; 2) aspirator dial at 6; 3) pressure at 1.5-2 Kgf/cm.sup.2 s; 4)
inlet temperature at 50.degree. C.; and 5) outlet temperature at
40.degree. C. The spraying took place under nitrogen for two hours. The
powder was collected and combined with amikacin drug solution (as prepared
in Example 1). The resulting solution was mixed for 2 minutes using a high
shear mixer (Virtishear)) at 1000 rpm. The solution was placed in a beaker
and set in a 40.degree. C. water bath and hydrated with mixing until the
solution reached 40.degree. C. (about 25 minutes). The solution was then
placed in a homogenizer (Gaulin 15M) for approximately 30 passes at 10,000
psi while maintaining the inlet temperature at 40.degree. C. The resulting
solution was filtered through a 0.8 micron nylon filter. The solution was
ultrafiltered to replace unencapsulated drug with new buffer. The solution
was washed with 7 to 10 volumes of buffer. The resulting product was
heated to 40.degree. C. and filtered through successive 0.8, 0.45, and
0.22 micron (pore size) filters. Thus, a surprising aspect of the present
invention is that the hydration of liposomes occurred significantly below
the transition temperature of the formulation (about 52.degree. C.).
EXAMPLE 3
The testing of liposome encapsulated amikacin for the treatment of MAC was
performed using a murine model. Beige mice (C57B1/6bg.sup.j /bg.sup.j)
were infected with MAC (101, type 1). The mice were infected by injection
of 1.times.10.sup.7 Colony Forming Units (cfu) in the mouse tail vein
(i.v.). Three experiments were performed. In the first experiment, 40, 80
and 120 milligrams of amikacin (liposomal and free) per kilogram of mouse
body-weight was given i.v. daily for 5 days, beginning 7 days after
infection. The animals were sacrificed 5 days after treatment was
completed and the liver, lung and spleen tissue were plated. Quantitation
of organisms in liver, spleen and lung tissue was performed. The cfu were
determined by growth on Middlebrook 7h11 agar plates. Untreated and empty
liposomes were used as controls.
The liposomes were prepared as in Example 1. This experiment was performed
in two identical parts. The liposomes in the first part were of an average
size of 49.8 nm and the average size of the liposomes in the second part
were 73.7 nm (mean diameter). The amikacin concentration was either 15.0
mg/ml or 13.21 mg/ml and the total lipid concentration was either 121
mg/ml or 55.7 mg/ml respectively (drug to lipid ratios: 0.123, 0.24). Free
drug for all experiments was prepared in the same buffer that the
liposomal formulations were contained in. The results are listed in Table
1. Table 1 lists the results for the spleen and liver tissue.
TABLE 1
______________________________________
Effects of Liposomal Amikacin on MAC Infected Mice Tissue
# Spleen Liver
Regimen mice cfu/g log cfu/g
cfu/g log cfu/g
______________________________________
untreated 6 1.13 .times. 10.sup.9
9.05 7.49 .times. 10.sup.8
8.87
liposomes only
3 5.30 .times. 10.sup.8
8.72 5.51 .times. 10.sup.8
8.74
amikacin 40 mg/
5 4.08 .times. 10.sup.8
8.61 1.41 .times. 10.sup.8
8.15
kg
amikacin 80 mg/
6 4.82 .times. 10.sup.8
8.68 7.79 .times. 10.sup.7
7.89
kg
amikacin 120 mg/
3 3.46 .times. 10.sup.8
8.54 5.65 .times. 10.sup.7
7.75
kg
liposomal 6 5.43 .times. 10.sup.7
7.73 1.44 .times. 10.sup.7
7.16
amikacin 40 mg/
kg
liposomal 6 2.71 .times. 10.sup.7
7.43 2.11 .times. 10.sup.7
7.32
amikacin 80 mg/
kg
liposomal 6 4.03 .times. 10.sup.7
7.61 9.86 .times. 10.sup.6
6.99
amikacin 120 mg/
kg
______________________________________
The results for the lung study were as follows: 1) untreated regimen were
2.86.times.10.sup.7 cfu/gram (log=7.46);2) empty liposomes were
2.95.times.10.sup.7 cfu/gram (log=7.47);3) free amikacin (40 mg/kg, 80
mg/kg and 120 mg/kg) were 1.44.times.10.sup.6 (log=6.16),
1.29.times.10.sup.6 (log 6.11) and 9.94.times.10.sup.5 (log=6.00)
respectively; 4) liposomal amikacin (40 mg/kg, 80 mg/kg, and 120 mg/kg)
were 2.28.times.10.sup.5 (log=5.36), 3.02.times.10.sup.5 (log=5.48) and
4.15.times.10.sup.5 (log=5.62).
Another experiment was carried out as above (also in two parts) except that
the drug therapy was started 5 days after infection. The drug was given
i.v. three times/week for 21 days. The mice were sacrificed 1-2 days after
the treatment stopped. During the treatment period, a group of mice
received 40 mg/Kg, 80 mg/kg, and 150 mg/kg of free amikacin and another
group received 40 mg/kg, 80 mg/kg, and 160 mg/kg of liposomal amikacin.
The lung tissues were not tested. Similar controls were used as above. The
liposomes were prepared as described in Example 1. The average size of the
liposomes was 66.5 nm or 79.6 nm (mean diameter). The concentration of the
amikacin was either 15.98 mg/ml or 6.74 mg/ml. The total lipid was measure
only the second part of the experiment and it was 27.0 mg/ml (drug to
lipid ratio: 1:4.167). The results are listed in Table 2.
TABLE 2
______________________________________
Effects of Liposomal Amikacin on MAC Infected Mice Tissue
Spleen Liver
Regimen # mice cfu/g log cfu
cfu/g log cfu
______________________________________
untreated 6 2.23 .times. 10.sup.9
9.35 1.06 .times. 10.sup.9
9.03
liposomes only
5 2.27 .times. 10.sup.9
9.36 2.08 .times. 10.sup.9
9.32
amikacin 40 mg/kg
6 5.76 .times. 10.sup.8
8.76 1.09 .times. 10.sup.8
8.04
amikacin 80 mg/kg
6 2.02 .times. 10.sup.8
8.31 3.67 .times. 10.sup.7
7.56
amikacin 120 mg/kg
2 1.48 .times. 10.sup.8
8.17 3.15 .times. 10.sup.7
7.50
liposomal amikacin
6 7.98 .times. 10.sup.7
7.90 7.24 .times. 10.sup.6
6.86
40 mg/kg
liposomal amikacin
4 2.57 .times. 10.sup.7
7.41 2.21 .times. 10.sup.6
6.34
80 mg/kg
liposomal amikacin
6 1.73 .times. 10.sup.7
7.24 1.04 .times. 10.sup.6
6.02
160 mg/kg
______________________________________
A third experiment was carried out as in the second experiment except that
the mice treated with the liposomal amikacin received 120 mg/kg, 240 mg/kg
and 320 mg/kg doses and the mice treated with the free amikacin received
only 120 mg/kg doses. The liposomes were prepared as in Example 2 where
the average size of the liposomes were 81.8 nm (median diameter). The
amikacin concentration was 32.02 mg/ml and the total lipid concentration
was 91.5 mg/ml (drug to lipid ratio: 1:2.941). The results are listed in
Table 3.
TABLE 3
__________________________________________________________________________
Effects of Liposomal Amikacin on MAC Infected Mice Tissue
Spleen Liver
Regimen # mice
cfu/g log cfu/g
cfu/g log cfu/g
__________________________________________________________________________
untreated
6 3.70 .times. 10.sup.9
9.57 1.65 .times. 10.sup.9
9.22
liposomes only
6 4.05 .times. 10.sup.8
8.61 1.20 .times. 10.sup.8
8.08
amikacin 120 mg/kg
6 2.13 .times. 10.sup.8
8.33 6.10 .times. 10.sup.7
7.78
liposomal amikacin
6 8.8 .times. 10.sup.6
6.94 3.67 .times. 10.sup.5
5.56
120 mg/kg
liposomal amikacin
6 5.52 .times. 10.sup.6
6.74 4.83 .times. 10.sup.5
5.68
240 mg/kg
liposomal amikacin
6 <3.38 .times. 10.sup.6
6.53 <2.00 .times. 10.sup.5
5.30
320 mg/kg
__________________________________________________________________________
The results of all the experiments establish that significantly higher
doses of amikacin can be delivered without an increase in toxicity and
with superior efficacy. It is known in the art that 150 mg/kg of amikacin
will kill many of the mice injected. Thus, the delivery of 320 mg/kg of
amikacin is a significant increase in the amount of drug delivered without
lethal toxicity.
EXAMPLE 4
The efficacy and toxicity of liposomal amikacin in Pseudomonas infected
mice was tested. CF-1 mice were used (females, 6-8 weeks old). The mice
were obtained from Jackson Labs. A clinical isolate of Pseudomonas
aeruginosa was obtained on a Mueller Hinton/MacConkey blood agar plate.
The organisms were transferred to a Mueller Hinton plate and grown for 24
hours. Colonies were transferred to saline and grown for 48 hours. The
samples were frozen in saline containing 15% fetal calf serum at
1.times.10.sup.8 organisms/ml (MacFarland Standard). The colonies were
stored at -70.degree. C. The colonies were thawed and grown in a Mueller
Hinton plate for 24 hours. The bacteria were adjusted to 8.times.10.sup.6
organisms/ml in saline containing talc (62.5 mg/ml) and 1 ml was injected
intraperitoneal. A culture of inoculum on MH agar for 24 hours determined
that approximately 7.times.10.sup.6 cfu were delivered per mouse.
Liposomal amikacin and empty liposomes were prepared as set out in Example
2. The liposomes of the amikacin formulation had an average size (median
diameter) of 62.4 nanometers. The lipid concentration was 95.31 mg/ml and
the amikacin concentration was 23.54 mg/ml (0.25 drug/lipid ratio). The pH
of the solution containing the formulation was 7.31. The empty liposomes
had an average size of 66.8 nanometers with 92.05 mg/ml of lipid. The
concentration of the free amikacin solution was 23.54 mg/ml. The pH of the
solution containing the liposomes was 7.33.
Mice were treated with 40 mg/kg (drug per body-weight), 80 mg/kg, 120 mg/kg
and 240 mg/kg of liposomal amikacin. Mice were also treated with 40 mg/kg,
80 mg/kg, and 120 mg/kg of free amikacin. The drugs were administered
intravenously in the caudal vein. Two doses were given; one at four hours
after infection and one at twenty-four hours after infection. The results
are listed in Table 4. The results establish that it is possible to
deliver up to 240 mg/kg of amikacin using a liposome formulation with all
the subjects surviving.
TABLE 4
______________________________________
Effects of Liposomal Amikacin on Pseudomonas Infection in Mice
Mortality
#died/#tested hours post infection
Infection
Treatment 24 30 48 72 %
______________________________________
no none 0/4 0/4 0/4 0
yes none 5/6 6/6 100
yes 40 (free amikacin)
1/4 1/4 1/4 1/4 25
yes 80 (free amikacin)
0/6 0/6 0/6 0/6 0
yes 120 (free amikacin)
0/6 0/6 0/6 0/6 0
yes 40 (lipo. amikacin)
1/4 3/4 4/4 4/4 100
yes 80 (lipo. amikacin)
0/6 0/6 1/6 2/6 33
yes 120 (lipo. amikacin)
0/6 0/6 0/6 0/6 0
yes 240 (lipo. amikacin)
0/6 0/6 0/6 0/6 0
______________________________________
EXAMPLE 5
Amikacin content was measured in mouse (C57B1/6, females 2-3 months old)
plasma after injection (i.v.) of free amikacin or liposomal amikacin (100
mg/kg). Liposomal amikacin was prepared as described in Example 2. Blood
samples were obtained at various time intervals. The samples were
collected by removing 100 microliters of blood, retroorbitally, from
nonanesthetized mice. The samples were collected in heparinized capillary
pipets which were plugged with modeling clay. The samples were centrifuged
at 3250 rpm (20 cm rotor) for 10 minutes. The samples were analyzed using
a radioimmunoassay (Diagnostic Products). The liposomal amikacin
formulation contained liposomes with an average size of 43.6 nm (median
diameter). The amikacin concentration was 10.09 mg/ml and the total lipid
concentration was 56.8 mg/ml (drug to lipid ratio 1:5.556). The results
are listed in Table 5.
TABLE 5
______________________________________
Amikacin Content in Mouse Plasma
Average Standard
Concentration
deviation
Treatment
Interval (hours)
# of mice
(.mu.g/ml)
(.+-..mu.g/ml)
______________________________________
Amikacin
0.0833 4 406 155
0.25 4 106 12
2 4 65 12
6 4 none detected
14 not tested
24 not tested
Liposomal
0.0833 4 530 258
Amikacin
0.25 4 344 321
2 3 346 199
6 4 278 137
14 4 272 78
24 6 108 14
______________________________________
The results establish that liposomal amikacin is retained in the plasma for
a significantly longer period than free amikacin.
Although this specification has been disclosed and illustrated with
reference to particular applications, the principles involved are
susceptible to numerous other applications which will be apparent to those
skilled in the art. The invention is, therefore, to be limited only as
indicated by the scope of the appended claims.
EXAMPLE 6
Liposome encapsulated amikacin was tested against both drug resistant and
drug susceptible strains of M. tuberculosis. Liposomes were prepared as in
Example 1.
Two test strains of M. tuberculosis were identified by the Gen-Pro (San
Diego) technique. One strain was a drug resistant strain known as
Vertulla. The other strain is known as H37RV. Both strains are located at
the National Jewish Center for Immunology and Respiratory Medicine,
Denver, Colo. For each experiment, a subculture in 7H9 broth was made from
a frozen aliquot. After 1 to 2 weeks of incubation at 37.degree. C. on a
constantly rotating roller drum, the bacterial suspension for each strain
was forced through a 27-gauge needle and then centrifuged at 250.times.g
for 5 minutes to remove large clumps of bacteria. Peripheral blood from
healthy purified protein derivative-negative donors was collected in a 60
ml syringe that had been pretreated with approximately 0.06 ml of a
preservative-free solution containing 10,000 IU of heparin per ml (GIBCO
Laboratories, Grand Island, N.Y.). This pretreated resulted in 10 IU/ml of
blood. The 60 ml blood sample was placed in a tube containing a
Ficoll-Hypaque gradient (Sigma Diagnostics, St. Louis, Mo.) and
centrifuged at 800.times.g for 15 minutes at room temperature, in
accordance with the manufacturer's instructions. The mononuclear cell band
was collected, transferred to a 50 ml Falcon tube, and diluted with RPMI
1640 (GIBCO), containing 10 IU of heparin per ml, to a total volume of
40.0 ml. The suspension was centrifuged at 250.times.g for 10 minutes at
room temperature. The cells were washed once in 10 ml of RPMI 1640
containing heparin and the pellet was resuspended in RPMI 1640 and
adjusted to 10.sup.7 cells per ml. The suspension was placed in 35 mm
plastic petri plates (Becton Dickinson Labware, Lincoln Park, N.J.) in two
spots, 1 drop (approximately 0.05 ml) for each, resulting in monolayers
containing about 5.times.10.sup.5 cells. The plates were incubated at
37.degree. C. for 1 hour to allow the cells to adhere and were then washed
twice with RPMI 1640. Then, 1.5 ml of RPMI 1640 containing 3% human
autologous nonheated fresh serum was added to each plate for a 7 day
period of incubation at 37.degree. C. in the presence of 7% CO.sub.2. The
pH of the medium was 7.2 to 7.3.
The above-described bacterial suspension was centrifuged at 3,500.times.g
for 30 min in a refrigerated centrifuge, and the pellet was resuspended in
2.0 ml of RPMI 1640. To estimate the number of acid-fast bacilli per
milliliter of this suspension, 0.01 ml samples of the suspension were
placed on Reich counting slides (Bellco Biotechnology, Vineland, N.J.)
with a known number of fields (under .times.1,000 magnification) per
circle. The slides were fixed and stained. The numbers of acid-fast
bacilli per milliliter estimated from the counts on these slides proved to
be accurate, as shown previously. On the basis of these counts, the
bacterial suspension in RPMI 1640 was adjusted to contain 10.sup.6
acid-fast bacilli per ml.
After 7 days of incubation, the monocytes were considered to have matured
into a macrophage monolayer. The medium was removed from the plates and
replaced with the bacterial suspension at 1.5 ml per plate. After
incubation for one hour, the plates were washed three times to remove the
extracellular bacteria. The infected macrophages were incubated in RPMI
1640 supplemented with 1% human autologous serum. Liposomal and free
amikacin was added to the cultures at the following concentrations
(.mu.g/ml of RPMI): 1, 2 and 4 for susceptible strain and 4, 8 and 16 for
resistant strain. Drug free liposomes and untreated cultures were used as
controls. The experiment was carried out in duplicate. The bacterial
counts used in the experiment described below represented only
intracellular bacteria. At days 0, 4, and 7, the medium from alternate
plates was discarded, and the monolayers were lysed by exposure for 10
minutes to a 0.25% solution of sodium dodecyl sulfate at 1.0 ml per plate.
After the suspension was transferred to a tube, the plate was rinsed with
1.0 ml of 7H9 broth containing 20% bovine albumin, and the rinse was then
added to the same tube. Tenfold serial dilutions were made to inoculate
7H11 agar plates for subsequent colony counts. The results were expressed
as the number of cfu per monolayer. The initial cfu (at day 0) is shown as
measured for the liposomes only (4.3.times.10.sup.3). ›The results of the
experiment are shown in Tables 6 &
TABLE 6
______________________________________
Activity of Amikacin (Liposomal and Free) Against Drug Resistant TB
(Vertulla)
0 4 7
Avg. Avg. Avg.
CFU log CFU log CFU log
DAYS (1 .times. 10.sup.3)
CFU (1 .times. 10.sup.3)
CFU (1 .times. 10.sup.3)
CFU
______________________________________
No Treatment 15, 30 4.4 500, 500
5.7
Liposomes
4.6, 4.0 3.6 10, 22 4.2 240, 360
5.5
Only
Liposomal
Amikacin
4 .mu.g/ml 1, 1.2 3.0 4, 3.5 3.6
8 .mu.g/ml 0.2, 0.5
2.5 0.28, 0.62
2.7
16 .mu.g/ml 0.05, 0.03
1.6 0.08, 0.09
1.9
Amikacin
4 .mu.g/ml 9.3, 8.0
3.9 210, 100
5.2
8 .mu.g/ml 5, 4 3.7 4.5, 6.2
3.7
16 .mu.g/ml 5, 5.4 3.7 3.7, 4.1
3.6
______________________________________
The results in Table 6 clearly indicate that liposomal amikacin provides an
effective means for treating drug resistant M. tuberculosis. The level of
activity against the growth of the drug resistant strain is a least about
8 times greater than free amikacin and provides activity up to at least
130 times greater than free amikacin at high doses (16 .mu.g/ml).
Liposomal amikacin at the doses studied obtained killing at each
concentration whereas the free amikacin obtained, at the most, inhibition.
TABLE 7
______________________________________
Activity of Amikacin (Liposomal and Free)
Against Drug Susceptible TB
0 4 7
Avg. Avg. Avg.
CFU log CFU log CFU log
DAYS (1 .times. 10.sup.3)
CFU (1 .times. 10.sup.3)
CFU (1 .times. 10.sup.3)
CFU
______________________________________
No 110, 200
5.2 1700, 1000
6.1
Treatment
Liposomes
8, 10 4.0 80, 90 4.9 1100, 1300
6.1
Only
Liposomal
Amikacin
1 .mu.g/ml 6, 7 3.8 100, 100
5.0
2 .mu.g/ml 2, 1 3.2 7.5, 8.6
3.9
4 .mu.g/ml 1, 1 3.0 2, 2.5 3.4
Amikacin
1 .mu.g/ml 50, 60 4.7 900, 800
5.9
2 .mu.g/ml 20, 20 4.3 40, 50 4.7
4 .mu.g/ml 4,5 3.7 1.5, 3 3.4
______________________________________
The results in Table 7 show that liposomal amikacin is as effective or
even more effective of inhibiting or killing M. tuberculosis at equivalen
doses.
The results in Table 7 show that liposomal amikacin is as effective or even
more effective of inhibiting or killing M. tuberculosis at equivalent
doses.
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